Unraveling of the whole human genome has shifted the focus of interest of scientists and researchers on the analysis of proteins; i.e., gene products. It may not be overstating to say that substantial protein analysis can be made possible only when a molecule that exhibits affinity (binding property) for each protein of interest has been successfully obtained. Cells, however, each contain quite many different types of proteins, and the amino acid sequence and structure of many of which are still unknown.
The most common technique for obtaining a molecule that exhibits affinity for a specific protein is to prepare an affinity antibody by utilizing the immune system of animal. However, this technique uses animals and thus, requires a large quantity of proteins, a large number of steps and large cost. Additionally, no affinity antibody can be obtained for specific substances with this technique.
A technique called the aptamer method (also referred to as the SELEX method) that does not rely on any living organism has been proposed to avoid this problem. However, while a molecule obtained by this technique strongly interacts with a specific protein, this technique is not applicable to all the proteins. In view of such circumstances, the present inventors proposed a modified aptamer method that is established by improving the aptamer method so as to use a modified nucleic acid (see International Publication No. WO2003/078623 pamphlet). However, since the modified aptamer method uses a modified nucleic acid having different types of substituents, the properties of each of the substituents have to be considered when amplifying a modified nucleic acid exhibiting affinity for a target protein. Thus, it has been difficult to find excellent PCR conditions. Additionally, the above method poses a problem that a functional molecule that tends to be strongly bound to a target substance is hard to be amplified by PCR.
In order to solve the above existing problems, the inventors have previously proposed dimer amidites—raw materials for modified nucleic acids to which substituents have been introduced or bound so that they can be removed through a treatment with ammonia, the substituents allowing binding to proteins. Here, the types of the substituents correspond one-to-one to the sequences of the dimer amidites; and the substituents are removed after binding to proteins and then, the resultant modified nucleic acid can be amplified by PCR. However, these dimer amidites each have both a moiety quite labile to an acid and a moiety quite labile to a base and thus, pose a problem in that they are decomposed to a considerable extent by purification. These dimer amidites, therefore, are forced to be used without purification. When certain dimer amidites are used, the synthesis yield of nucleic acid using an automatic nucleic acid synthesizer may be lowered. Thus, further improvement is demanded.
Meanwhile, a solid-phase synthesis of nucleic acid has been performed for 20 years or longer, and an automatic synthesizer employing it was also sold at that time. The solid-phase synthesis of nucleic acid is performed by, for example, condensating nucleic acid raw materials (amidites) with nucleosides bound to a solid-phase support (e.g., CPG). During this condensation reaction, it is necessary that only the phosphoric acid moiety of each amidite is condensated with only the hydroxyl group of another amidite so that the other reactive groups do not participate in the condensation reaction. Thus, protective groups are introduced to the reactive groups (e.g., exocyclic amino groups of bases of amidites used and a phosphoric acid moiety which is not made to participate in the condensation reaction) so that they do not participate in the condensation reaction, and the protective groups are removed (deprotected) after completion of the whole condensation reaction. Conventionally, a benzoyl group, an isobutyryl group, other groups have been used as a protective group which is introduced to the exocyclic amino group of a base, and these protective groups are generally removed by treating the obtained nucleic acid with concentrated aqueous ammonia at 55° C. for 8 hours to 15 hours.
However, in the production of the above-described modified nucleic acids having affinity (binding property) for proteins, under such conventional deprotection conditions, not only the protective groups but also their modified moieties (substituents having binding property for proteins) are removed, resulting in that modified nucleic acids cannot be stably produced. Thus, in the production of such modified nucleic acids, in order to prevent the substituents having binding property for proteins from being removed together with the protective groups, there is a need to use amidites having protective groups which can be removed under milder conditions.
For example, some conventional literatures report nucleic acid amidites having protective groups which can be removed by diazabicycloundecene (DBU) (i.e., a bulky base) (Acta. Chem., Scand., B37, 263 (1983) and J. Org. Chem., 54, 1657 (1989)). But, these nucleic acid synthesizing amidites are not stable in acetonitrile (i.e., an aprotic solvent) (Tetrahedron Letters No. 46, 6729 (1990)) and are not suitable to practical use. Other literatures report nucleic acid synthesizing amidites having protective groups which can be removed in pyridine using 0.5M DBU for 16 hours (Tetrahedron No. 20, 4171 (1992) and Nucleodied & Nuclrotides 13, 2059 (1994)). But, the use of a high concentration of DBU and the deprotection for a long time problematically cause alkylation of the base of nucleic acid. Other literatures report nucleic acid synthesizing amidites having protective groups which can be removed in methanol using K2CO3 (Tetrahedron Letters No. 46, 6729 (1990) and Nucleic Acids Reserch 21, 3493 (1993)). But, use of K2CO3 (a base) in methanol (a protic solvent) problematically causes decomposition of the esters, etc.
Under such circumstances, at present, demand has arisen for developments of a nucleic acid synthesizing dimer amidite which can be subjected to purification, preferably, whose protective groups can be removed under mild conditions; and a nucleic acid synthesizing method using the nucleic acid synthesizing dimer amidite.